Transfer of gene conferring herbicide bialaphos resistance into buckwheat plants

Bar gene conferring resistance to herbicide bialaphos (phoiphinothricin) was cloned from Streptomyces hygroscopicus. Bar gene under 35S promoter of cauliflower mosaic virus was introduced in binary pBinl9 vector and constructed p las mid was transferred into Agrc bacterium tumefaciens strain. Conditions of genetic transformation of cultivated buckwheat and interspecific hybrid Fagopirum esculentum * F. tataricum were worked out Buckwheat explants were inoculated by the strain with a plasmid carrying bar gene nearby NPT11 gene. Molecular analysis of 8 regenerated plants that were selected for kanamycin resistance was performed. 5 plants gave positive signal by doiand Southern hybridization using as a probe DNA fragment with bar gene that is an evidence of integration of bar gene into plant genome. Transformed plants grow and rooted at Basta concentrations in the mediut і that totally inhibited nontmnsformed plants.

Introduction.The implementation of herbicides in modern agriculture is inevitable [1 ].They control the weed growth and thus increase harvest [2].Use of herbicides of a new generation that are very effective and as a rule inhibit single definite enzyme at very low concentration is very attractive.These herbicides are not toxic for human and animals.The ecological safety is connected to their quick degradation in soil and absence of target enzymes in vertebrates [1,3].
Bialaphos is an example of a herbicide of this generation.Tt is a tripeptide and its acting substance is phosphinothricin (PPT) a toxic analog of glutamic acid [4].But bialaphos as many other herbicides of this generation is not selective, inhibiting the growth of any plant and this character diminished the use of these herbicides in agriculture.Using gene engi neering approach by introducing into plant genome of gene(s) conferring resistance to herbicides of this type it is possible to produce transgenic plants resis tant to these herbicides [5].
The source of these genes is soil and other microorganisms.Bialaphos resistance gene (bar) was found in the genome of bialaphos producing strep tomyces , conferring their resistance to produced tri peptide [4].
Production of transgenic tobacco, potato, tomato, aspen plants resistant to herbicide PPT is reported II,3,6,7].Earlier we described the introduction of modified aroA geie conferring resistance to herbicide glyphosate (Roundup) into potato, sugarbeet and soybean plants [J!, 9].In this paper we report about transfer of bar gene and its expression in buckwheat plants.
Alcali plasmid isolation, restriction, DNA ligation and electrophoresis, DNA elution and its cleaning, transformation of bacteria were done by the described methods [12,13].The binary vectors were trans ferred by tri-parental matings to the disarmed Agrobacterium tumefaciens strains 3850 [12] and LB A 4404.
All the enzymes used in this work were obtained from NPK «Biotekh» (Russia).
In all experiments we used mutant form of buckwheat Homo;$tilnaya with high regeneration po tential and hybrid of this form with tartari buckwheat (k~17).This hybrid was obtained via in vitro culture of immature interspecific embryos [14].
Internodes, petioles and leaves of these hybrids and Homostilnaya were inoculated by 3850 strain of A. tumefaciens by two hours co-cultivation in over night culture of agrobacteria in the presence of acetosyringone.Then explants were transfexred onto Murashige-Skoog basal media with the addition of 1 mg/l BA and 0.5 mg/I NA for plant regeneration.To suppress agrobacteria regrowth and for kanaraycin selection (NPTII gene is located on plasmids along side bar gene) antibiotics kanamycin ?carbenicillin and claforan were also added to the culture medium.Explants were cultivated first in the dark and after appearence of regenerants under 16:8-hr photoperiod with constant temperature of 26 °С.Regenerants that came through kanamycin selection were rooted and microclonally propagated.For evaluation of their herbicide resistance these regenerants were trans ferred onto MS medium with 3-30 jul/\ concen trations of PPT (Basta).
Results and Discussion.It was shown that recom binant pBA-1 plasmid conferring bialaphos resistance has additional L7 kb pair Pst fragment.To improve bar gene expression GTG translation initiation codon was changed for eukaryotic ATG codon.
35S promoter of cauliflower mosaic virus and 3' region from nopalin synthase gene were introduced into bar gene expression cassette.Recombinant pBA-3 plasmid was constructed for transfer of bar gene into buckwheat plants using agrobacteria transformation system.For this purpose Hind III fragment containing 35S promoter, bar gene and poly A region of nopalin synthase gene was cloned into corresponding site of pBinl9 binary vector.
Plants regenerated on kanamycin containing me dium that was changed every 12-14 days were analyzed by dot-and Southern blot-hybridization.Out of 8 kanamycin resistant regenerants positive autoradiographic response gave 5 plants: 4 plants of hybrid Homostilnaya* к-17 and one plant of Homostilnaya that is a proof of bar gene integration into plant's genome (Fig. 1,2).
These 5 plants were microclonally propagated and transferred into MS medium containing 3, 10, 15, 20 and 30 ui/l of herbicide Basta.On medium added with 3 and 10 ul/l of Basta all these plants formed roots and their growth did not differentiate from the growth of plants on MS medium without herbicide.
At 15 and 20 //1/1 of Basta only 2 plants rooted and their development was normal.At 30 Basta concentration only one plant survived.The growth and root formation of nontransformed plants of Ho mo stilnaya and Homostilnayax k-17 hybrid were in hibited already at 3 /ul/І of Basta.That is a difference among transformed plants by the level of traisferred bar gene expression was clearly seen.

Fig
Fig. 1.DNA dot-hytridization of buckwheat plants Arrows show negative (/) and positive (2) controls.Intensive black spots cor respond to DNA of phnts with positive autoradiographic response Fig. 2. DNA Southern hybridization of buckwheat plants: Уpositive control (Hindi!J fragment with bar-gene); 2-6 -DNA of buckwheat plants aft;r Hindlll restriction with positive autora diographic response; 7 -negative control (DNA of nontransgenic buckwheat plants)